Article Figures & Data
Figures
- Figure 1.
SNCG renders a drug resistance. A, effect of SNCG on docetaxel resistance in breast cancer cells. SNCG-negative MCF-7 and MCF-neo1 (vector transfect clone), SNCG stably transfected MCF-S2 and MCF-S6 cells (top); SNCG-negative MDA-MB-435 and neo-435-1 (vector-transfected clone), and SNCG stably transfected SNCG-435-3 cells (bottom) were treated with or without 100 nmol/L docetaxel for 2 days and apoptotic cells were determined. The numbers represent means ± SD of three cultures. Statistical comparisons of treated versus control indicate P < 0.001. B, effects on apoptotic biomarkers. MCF-7 and MCF-S6 cells were treated with or without docetaxel (100 nmol/L, 48 hours). Equal amount of total cellular protein was subjected to Western blot analyses of PARP, cleaved PARP, and actin. C and D, effects of knockdown SNCG expression on SKBR-3 (C) and MDA-MB-231 (D) cells on docetaxel resistance. Inserts indicate the reduced SNCG expression in the siRNA knockdown cells. Cells were treated and analyzed as described for MCF-7 and MDA-MB-435 cells. E, effects of SNCG on nocodazole resistance. MDA-MB-435 and SNCG-435-3 cells were treated with nocodazole (0.5 μmol/L, 30 hours) followed by analysis of apoptosis. F and H, SNCG renders tumor resistance to docetaxel. Tumor growths of MDA-MB-435 (F) and MCF-7 (H) xenografts in response to docetaxel treatment. Tumor cell injection and estrogen supplement (for MCF-7 cells) have been described in Materials and Methods. Mice bearing established tumors were treated with either vehicle control or docetaxel (25 mg/kg, i.p., one time per 5 days for 25 days). All mice were sacrificed at day 25 following the first drug treatment. Statistical comparison for tumor sizes in docetaxel-treated SNCG-negative MDA-MD-435 and 435-neo-1 mice relative to mice in other groups indicates P < 0.01. Statistical comparison for tumor sizes in docetaxel-treated MCF-7 mice relative to mice in other groups indicates P < 0.005. G and I, effects of SNCG on apoptosis of MDA-MB-435 (G) and MCF-7 (I) tumor xenografts. Histologic sections of tumors from docetaxel-treated or control mice were analyzed for apoptosis (TUNEL assay). Statistical analysis showed that docetaxel-treated SNCG-expressing tumor tissues had a significantly lower percentage of TUNEL-positive cells than did docetaxel-treated SNCG-negative tumor tissues (P < 0.01).
- Figure 2.
Structure analyses of the in silico interaction between BubR1 with C-terminal region of SNCG. A, docked interacting conformation of BubR1 [determined by X-ray crystallography (36)] with the in silico predicted structure of SNCG-C terminal region (blue). A1 and A2, the interaction mode between the residues of BubR1 (yellow) involved in binding with SNCG (sky blue), which influence the displacement in the GIG motif-1 (A1) and those that cause the structural transition 1 (A2). B, structural superimposition of native BubR1 X-ray crystallographic structure (green) with the structure obtained after docking with C-terminal region of SNCG (chocolate brown). C, displacement in the position of GIG motif of BubR1 (green) compared with that of native BubR1 structure (red). D, structural change occurred in the BubR1 [Gln110-Pro119; blue] compared with that of native BubR1 structure (green). E, structural displacement occurred in the region [Pro74-Ala108] of BubR1 (violet purple) with native BubR1 structure (lemon green).
- Figure 3.
Effects of SNCG C-terminal on interaction with BubR1 and drug resistance. MCF-7 cells, engineered to express either full-length F-SNCG or C-terminal–deleted D-SNCG (AA 106–127 deleted), were selected for stable clones. A, effects on interaction with BubR1. F-SNCG- and D-SNCG–transfected MCF-7 cells were treated with docetaxel (80 nmol/L, 20 hours), extracts were immunoprecipitated with polyclonal SNCG antibody, and blotted with BubR1 antibody. Total cellular (before immunoprecipitation) proteins of SNCG, BubR1, and actin were also analyzed as inputs. B, in vitro effect of SNCG on docetaxel resistance. SNCG-negative parental MCF-7 and neo-transfected control MCF-neo1 cells (vector-transfected clone), F-SNCG stably transfected MCF-S6 cells, and D-SNCG stably transfected MCF-D2 and MCF-D6 cells were treated with or without 100 nmol/L docetaxel for 2 days and apoptotic cells were determined. The numbers represent means ± SD of three cultures. Statistical comparisons of treated versus control indicate P < 0.001. C, in vivo effect of SNCG on docetaxel resistance in tumor xenograft model. Tumor cell injection and estrogen supplement have been described in Materials and Methods. Mice bearing established MCF-7, MCF-S6, and MCF-D2 tumors were treated with either vehicle control or docetaxel (25 mg/kg, i.p., one time per 5-days for 25 days). All mice were sacrificed at day 25 following the first drug treatment. Statistical comparison for tumor sizes in docetaxel-treated SNCG-negative MCF-7 and D-SNCG stably transfected MCF-D2 mice relative to mice in other groups indicates P < 0.01. IP, immunuprecipitation.
- Figure 4.
SNCG binds to BubR1, compromises BubR1 interaction with Cdc20 and Mad2, and inhibits BubR1 kinase activity. A and B, effects of SNCG on BubR1′s interaction with Cdc20 and Mad2 in MCF-7 (A) and MDA-MB-231 (B) cells. MCF-7 (parental and MCF-S6) and MDA-MB-231 (control siRNA and SNCG siRNA) cells were treated with docetaxel (80 nmol/L, 20 hours) and extracts were immunoprecipitated with SNCG, Cdc20, and Mad2 and blotted with BubR1 antibody. Total cellular (before immunoprecipitation) proteins of BubR1, SNCG, Cdc20, and actin were also analyzed as inputs. C, MCF-7 cells, engineered to express full-length F-SNCG and C-terminal–deleted D-SNCG (AA 106–127 deleted), were treated with docetaxel (80 nmol/L, 20 hours) and extracts were immunoprecipitated with BubR1 and blotted with Cdc20 antibody. D, expression of SNCG in MCF-7 cells correlates with reduced SAC kinase activity. Levels of phospho-BubR1 (P) and phospho-histone H3 (p-his3; representing SAC kinase activity in vivo) were determined by Western blotting on extracts from MCF-7 and MCF-S6 cells treated with or without docetaxel (80 nmol/L. 20 hours). E, SNCG inhibits BubR1 kinase activity in vitro. MCF-7 and MCF-S6 cells were treated with 80 nmol/L docetaxel for 20 hours. Endogenous BubR1 was immunoprecipitated from docetaxel-arrested cells and immunoblotted with anti-BubR1 antibody or kinase activity assayed after addition of histone H1 and [32P]ATP. F, overexpression of BubR1 restored the mitotic checkpoint control. MCF-neo1 and MCF-S6 cells were transfected with pCS2-BubR1 or control vector. At 30 hours posttransfection, cells were treated with DMSO or 80 nmol/L docetaxel for 20 hours. For each cell line, 200 to 300 cells randomly chosen from five different views were scored for mitotic arrest based on MPM-2 immunoreactivity. We examined the status of phosphoproteins that are specifically recognized by MPM-2 antibody and found only in mitotic phase cells using flow cytometer. Data are expressed as percentage of MPM-2–reactive cells in the propidium iodide (PI)–positive population. The numbers represent means ± SD of three cultures. A representative experiment is shown. IP, immunoprecipitation.
- Figure 5.
SNCG renders lower mitotic index. Cells from MCF-7 (A), MDA-MB-435 (B), SKBR3 (C), and MDA-MB-231 (D) were cultured in cover slips inserted into wells of 24-well culture plates and treated with DMSO or 80 nmol/L docetaxel for 20 hours. Data are expressed as percentage of MPM-2–reactive cells in the propidium iodide (PI)–positive population as described in Fig. 4. The numbers represent means ± SD of three cultures.
- Figure 6.
A and B, representative SNCG immunohistochemical stainings for SNCG- and SNCG+++ tumors. Sections were immunohistochemically stained with specific anti-SNCG antibody with brown color indicating SNCG protein expression in breast cancer cells. All sections were also counterstained lightly with hematoxylin for viewing non-SNCG-stained cells. A serial slide from the same block was also incubated with nonimmunized control immunoglobulin G (IgG), and no detectable background staining was observed. C, individual changes in apoptotic index at pretreatment and posttreatment of docetaxel and epirubine for patients with SNCG-negative and SNGC-positive tumors.
Tables
- Table 1.
Cytotoxicity of docetaxel
Cell lines SNCG expression status Cytotoxicity IC50 MCF-7 Negative 27 MCF-S6 Transfected 89 MDA-MB-435 Negative 63 SNCG-435-3 Transfected 153 T47D Positive 68 AS-3 Knockdown 29 NOTE: Stable SNCG-transfected MCF-7 (MCF-S6), MDA-MB-435 (SNCG-435-3), and SNCG knockdown T47D (AS-3) cells were previously established. All six cell lines were treated with docetaxel (ranging from 10 to 250 nmol/L) for 24 hours. Cytotoxicity was measured by the MTT assay. The IC50 values were defined as the concentration of drug eliciting 50% cell killing and determined by regression analysis.
- Table 2.
Changes of tumor size, Ki67 expression, and apoptotic index
Prechemotherapy Postchemotherapy t P Size (cm) SNCG+ 5.94 ± 1.76 4.17 ± 1.10 5.97 0.000 SNCG− 6.17 ± 1.64 3.00 ± 0.95 7.82 0.000 t −0.347 3.000 P 0.731 0.006 Ki67 (%) SNCG+ 26.67 ± 18.55 20.56 ± 11.49 0.09 0.052 SNCG− 32.50 ± 14.85 14.17 ± 9.73 4.26 0.001 t −0.910 1.582 P 0.370 0.125 Apoptotic index (%) SNCG+ 8.17 ± 6.64 10.56 ± 8.12 −0.84 0.411 SNCG− 11.83 ± 8.79 25.92 ± 19.62 −2.69 0.021 t −1.301 −2.569 P 0.204 0.023
Volume 13, Issue 3
